1. Field of the Invention
The present invention relates to an immunostimulatory compositions comprising liposome-encapsulated oligonucleotide and epitopes.
2. Description of the Related Art
Epitope-based peptide vaccines are extensively studied that are pivotal for inducing and regulating immune responses through their binding ability to B cell receptor (BCR) and MHC as B-cell epitope and T-cell epitope for protecting infectious and malignant diseases (1-3). Chemically inactivated vaccines are widely used in the clinics, however, the vaccines have disadvantages, such as the risk of virus reactivation, the cost for the maintenance of the vaccine stability, leading to autoimmune diseases, not support to sufficient protection in some of vaccines (1, 3, 4). For overcoming the theses disadvantages, synthetic peptides were developed for last 30 years to manipulate of the immune responses through the use of epitopes designed for stimulating particular subsets of lymphocytes, leading to selective B- and T-cell responses. Therefore, peptide vaccines have gained attention as potentially useful prophylaxis and therapy for anticancer vaccines (5, 6) and infectious diseases such as influenza virus (3), malaria (7), hepatitis B (8), and HIV (9). Although peptide vaccines were actively studied in various animal models, their efficacy is limited to treat human. To improve the peptide vaccine efficacy, liposomes are evaluated for delivery of vaccines (10) and adjuvants such as flagella (11) and CpG-DNA (12) were formulated for enhancing the magnitude of immune responses.
Liposomes as vehicles for delivery have been extensively evaluated in developing vaccines to enhance cytotoxic T lymphocytes (CTL) responses (10, 13). Encapsulated liposomes can protect from environment and deliver to target cells. pH-sensitive liposomes such as phosphatidyl-β-oleoyl-γ-palmitoyl ethanolamine (DOPE, dioleryl phosphatidylethanolamine)/cholesteryl hemisuccinate (CHEMS) are characteristics release of contents into the cytosol and lipid intermixing at low pH (5.0) (14). Investigators have shown that the pH-sensitive liposomes improve antigen delivery to the cytosol and induction of CTL responses (15). Furthermore, effective antigen specific CTL responses are reported in mice immunized with CTL epitopes synthesized from Hantaan nucleocapsid protein (M6) or human papilloma virus E7 encapsulated in pH-sensitive liposomes (16). To improve uptake of antigens by macrophages and dendritic cells, cationic liposomes were used as vehicles for delivery. The CTL response and antibody production are enhanced by encapsulated cationic liposomes such as lipofectamine, DC-Chol, DC-Chol/DOPE, EPC/SA/C etc (10).
Although liposomes are potent vehicles for delivering antigen to APCs, it is investigated to enhance of immunogenicity and adjuvanticity. The immunostimulatory activities of CpG-DNA have gained attention as a potentially useful form of therapeutics for immunoadjuvants compare to other immune-stimulating agents such as flagella, lipid A, cytokines etc (17, 18). Several investigators have shown that CpG-DNA upregulates antigen-presenting cell activity, Th1 immune response, immunoglobulin (Ig) isotype switching (19-21). The immunostimulatory activities as a potent adjuvant are enhanced by liposome-encapsulated CpG-DNA. Suzuki et al., show that CpG-DNA encapsulated in cationic liposomes induces expression of IL-12 and IFN-γ and CpG-DNA-liposome coencapsulated with ovalbumin (OVA) caused the induction of OVA-specific CTLs, which exhibited potent cytotoxicity against OVA-expressing tumor (22). In addition, SSCL improves the uptake by B cells, dendritic cells, and macrophages and coencapsulation of CpG-DNA with OVA magnified the Ag-specific IFN-γ and IgG production (23). Furthermore, Li et al., investigate that CpG-DNA and HER-2/neu-derived peptide coencapsulated in DSPC/Chol liposomes enhances the CTL response and IgG production (24).
Phosphorothioate-modified CpG-DNAs (PS-DNA), which is a sulfur substitution for the nonbridging oxygens in the backbone providing its nuclease resistance and efficient uptake into cells, have been used for clinical applications (25). However, several studies indicated that PS-DNA induces backbone-related side effects such as transient splenomegaly, lymphoid follicle destruction, and arthritis (26-28). Therefore, investigators developed the natural counterpart of the phosphodiester bond CpG-DNA (PO-DNA) to induce optimal innate immune responses without severe side effects. In contrast to PS-DNA, effects of PO-DNA were not seen in human cells. However, inductions of effective immune responses are reported in human cells stimulated with PO-DNA and non-CpG-DNA encapsulated in liposomes (DOTAP, lipofectin) (29, 30).
In previous studies, we identified the natural phosphodiester bond CpG-DNA (PO-DNA) through computer-assisted analysis of M. bovis genomic DNA and screened the genomic DNA sequences of M. bovis with immunostimulatory activity (31). Our experimental analyses demonstrate that a potent PO-DNA, namely MB-ODN 4531(O), containing CpG motifs has functional effects as a powerful adjuvant for the induction of Ag-driven Th1 responses without severe side effects (31, 32). In this study, we compared the ability of the PO-DNA (MB-ODN 4531(O)) encapsulated in several liposomes to stimulate immune responses in human and mice cells. Further, we show that PO-DNA (MB-ODN 4531(O)) and several peptides coencapsulated in DOPE/CHEMS liposomes significantly enhanced the peptide-specific IgG production. These results suggest that peptide vaccine efficacy were improved by delivery with DOPE/CHEMS liposomes and adjuvants effect with MB-ODN 4531(O), which can be promptly used for application in pandemic of infectious diseases, development of therapeutic antibody, and exposure of bioterrorism agents.
Throughout this application, various publications and patents are referred and citations are provided in parentheses. The disclosures of these publications and patents in their entities are hereby incorporated by references into this application in order to fully describe this invention and the state of the art to which this invention pertains.